Utilizing the human fibroblast model of cellular aging we will test the hypothesis that biological aging is a process similar to cell differentiation and accompanied, therefore, by alterations in genetic organization and/or expression. We will seek these alterations in several cell strains at intervals of their limited replicative lifespan derived from normal donors of chronological ages ranging from fetal to elderly, from subjects with inherited syndromes of premature aging, and SV-40 transformed fibroblasts and HeLa cells. Thus, we can compare gene organization and expression in cells undergoing normal and rapid aging and "immortal" cell sthat have "escaped" from senescence. Using specific pure hybridization probes and DNA restriction endonucleases we will study four levels of molecular organization wherein the activity of certain genes may be regulated. I. DNA Primary Structure. We will look for deletions or rearragements in and around: 1. Unique copy genes that are (a) normally expressed and essential for human fibroblasts, e.g. Beta-actin, (b) nonexpressed and nonessential, e.g. Alpha-globin or (c) expressed but nonessential, e.g., HGPRT. 2. Reiterated copy sequences, e.g., Eco-RI, Alu-I and Ul repeats. 3. Mitochondrial DNA. II. DNA Methylation. We will quantify the extent and stability of DNA methylation in and around these unique and reiterated genes. III. Chromatin. We will determine DNaseI sensitivity of these genes in nuclear preparations to learn whether the chromatin exists in an "open" or "closed" transcriptional state. IV. Gene Transcription. We will measure RNA copy number of these genes and study labeling and pulse-chase kinetics to determine rates of RNA transcription, processing and turnover. Correlations will be sought between the rate of gene transcription, the degree of methylation and the level of DNaseI sensitivity. These studies may reveal specific alterations in gene organization and expression that can be implicated in aging. They may also enhance our understanding of how certain mutant genes lead to accelerated aging and heightened predispostion to age-dependent diseases such as atherosclerosis and cancer.